We are developing chemical and physical probes for the study of the mechanism of thiamine pyrophosphate and Mg(11) catalyzed reactions. Our attempts initially are directed to the yeast enzyme pyruvate decarboxylase. The proposed work includes: a. T 1 and chemical shift nmr determination of the binding geometry. b. Replacement of the Mg(11) ion by various metals which can act as reporter groups of the active site environment. c. Model studies on the coenzyme binding site and the solvemt dependence of such binding phenomena. d. Synthetic modifications of the coenzyme and substrate to act as reporter and affinity labels. e. Determination of C12/C13 kinetic isotope effect of the enzyme reaction to pinpoint rate limiting step.